Project 5: In addition to adhering to each other and to the vessel wall, platelets contribute to thrombotic events by releasing bioactive molecules such as ADP, TxA2 and CD40L. In studies that form the basis for this proposal, human and mouse platelets were found to express on their surface the class IV semaphorin, sema4D or CD 100, a protein best known for its role in B-cell/T-cell interactions. It was also found that activated platelets shed the exodomain of sema4D and the "sheddase" was identified as the TNFa cleaving enzyme, ADAM17. Based on these observations and preliminary studies on the effects of soluble sema4D on platelets, we have developed the following hypotheses: 1) sema4D, either as a soluble molecule or surfacebound, contributes to platelet activation by binding to receptors expressed on nearby platelets, 2) plateletderived sema4D can also affect cells other than platelets within the circulation and the vessel wall, and 3) plasma levels of soluble sema4D will increase when pathological platelet activation occurs. To test these hypotheses, Aim #1 will examine the role of sema4D in platelet activation. Aim #2 will investigate the regulated shedding of the sema4D extracellular domain. Aim #3 will examine the role of CD72 and plexin- Bl as candidate receptors for platelet-derived sema4D in platelets, monocytes and endothelial cells, and Aim #4 will ask whether platelet activation in vivo causes a measurable increase in plasma sema4D levels that correlates with the extent of platelet activation. Aims #1-3 will take advantage of existing mouse lines lacking sema4D, CD72 or ADAM17. Aim #4 will make use of samples from two clinical trials in which platelet activation is expected. The first trial includes the 1,000 patients undergoing cardiopulmonary bypass in the prospective heparin-induced thrombocytopenia (HIT) trial that is part of Project #1. All of these individuals should have transient platelet activation while on bypass. Those that develop HIT will have persistent platelet activation. The second trial includes 4,000 patients with well-characterized atherosclerotic cardiovascular disease, a setting where platelet activation is predicted to occur, but be less pronounced. Collectively, these aims will address the basic biology of platelet sema4D and its receptors, explore the consequences of sema4D release when platelets are activated, and begin to assess the role of soluble sema4D as a contributor to thrombotic events in vivo.